HL-1 cells express an inwardly rectifying K+ current activated via muscarinic receptors comparable to that in mouse atrial myocytes

Abstract

An inwardly rectifying K(+) current is present in atrial cardiac myocytes that is activated by acetylcholine (I(KACh)). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gbetagamma G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPgammaS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.

Fig. 1

K⁺ current activated by carbachol in HL-1 cells. Carbachol activates an inwardly rectifying K⁺ current in HL-1 cells. Sample voltage-clamp recordings and current voltage relationships are shown

Fig. 2

K⁺ current activated by carbachol in HEK293 stable cell line. Carbachol activates an inwardly rectifying K⁺ current in a stable HEK293 cell line expressing the M2 receptor and the GIRK1 and GIRK4 channel subunits. Sample voltage-clamp recordings and current voltage relationships are shown

Fig. 3

Dependence of K⁺ current activation on G-proteins in HL-1 cells. a The bar charts summarise the results obtained. Treatment with pertussis toxin (100 ng/ml for 16 h) or tertiapin-Q (100 nM) led to inhibition of the carbachol-activated current (p < 0.01 for both). b Carbachol (10 µM) was applied four times for 1 min, with 1-min interval between applications, in the presence or absence of 0.3 mM GTP. Decline of the current elicited by carbachol was obtained only in the set of experiments where GTP was omitted (p < 0.05). c Currents were recorded for 3 min after break-in before addition of 10 µM carbachol in the presence of 30 µM intracellular GTPγS. Currents activated with dialysis of GTPγS into the cell (p < 0.05 compared with current at t = 0) and carbachol evoked smaller currents

Fig. 4

Kinetics of activation of the K⁺ current. Representative current traces recorded from HL-1 cells, native mouse atrial myocytes and the M2 GIRK1/4 cell line. Cells were clamped at −80 mV and 10 µM carbachol was applied for 20 s. The mean parameters are summarised in the Table. Dotted lines indicate zero current

Fig. 5

Immunostaining of HL-1 cells. Immunofluorescent staining of HL-1 cells and the M2 GIRK1/4 cell line stained for GIRK4 and the M2 receptor, respectively. Arrows show potential membrane localisation. In the lower panels of the figure, two controls are shown. In parental HEK293 cells without GIRK4 expression, the GIRK4 antibody does not stain the cells. Incubation of the M2 GIRK1/4 cell line with only the secondary antibody and not the primary does not lead to staining

Fig. 6

Quantitative real-time RT-PCR. ∆Ct values in quantitative real-time RT-PCR experiments performed as indicated in the methods. Data are shown as mean ± SD (n = 3 for HL-1 cells and n = 6 from two mice)